The change of the low end signals when starting to vibrate
the flow cell usually comes from the fact that you may hit
the liquid stream at a point were the surface starts to
ripple.
Supravital counterstain is ethidium bromide (5ug/ml) in PBS
with 0.1% azide and 0.05% Tween 20. It can be used with
both, PE and FITC labeled antibodies. For our studies of
dental bacteria we usually used a triple staining of FITC
antibody, EB supravital and PI dead cell label to sort
intact bacteria. Alternatively CFDA, PE-antibody and PI can
be used.
The azide is required to deenergize the pumps that otherwise
pump out the supravital stains. In a lot of cases this can
as well be achieved using a TRIS/EDTA buffer.
Triggering works best with peak scatter in the Elite, but I
do not know about the timing issues on the FACS star. If you
stay tuned on scatter, you know at least what else has been
there. If possible trigger on both, scatter and DNA
fluorescence.
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: Sorting Bacteria
Author: BarrenP@MedImmune.com at INTERNET
Date: 05/04/97 05:22
Flowers!
Does anyone have any experience with sorting bacteria (Streptococcus)
on a stream in air flow cytometer. I am using a
FACSTAR Plus.
What I think I need is a dye that will allow me to counter stain all
the bacteria (preferably live) to be used in conjunction with a dim
surface label, and then sort on the surface labeled positives. The
counter stain would need to be dim so as not to interfere with the dim
surface label.
I have tried to sort with a SSC log threshold. and this does not work.
As soon as I vibrate the stream I loose all low end resolution which
is where my bacteria are.
I need to use a fluorescent trigger to threshold on, this would be less
sensitive to stream vibrations which is what I assume is killing me on
the log SSC threshold.
Just a note all my Scatter parameters are on logarithmic amplification.
Thanks in advance for your time and energy
Philip Barren
Pb
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